Transplantation of vascularized allografts is an effective therapeutic alternative for patients with end-stage organ failure. Transplantation of organs between genetically different individuals, however, is limited by our ability to control the immunological rejection of the graft by the recipient. Immunosuppressive drugs may reduce the severity of rejection, but fail to create a state of permanent specific tolerance to the graft. We herein propose an immunomodulatory approach using a modified form of FasL with potent apoptotic activity to eliminate alloreactive T cells for the prevention of cardiac allograft rejection and induction of tolerance. FasL-induced apoptosis is the main mechanism of activation-induced cell death that is responsible for immune homeostasis and self-tolerance. The use of wild type (wt)FasL to prevent allograft rejection has been controversial. FasL is initially synthesized as a membranous molecule that induces apoptosis when prevention the cell surface. wtFasL is also shed from the cell surface by metalloproteinases within minutes of expression and the soluble form is antiapoptotic and chemotactic for neutrophils. We hypothesize that FasL can be used as an immunomodulatory molecule if its apoptotic activity is separated from anti-apoptotic and chemotactic functions. We generated modified forms of FasL with potent apoptotic activity and developed a novel approach to express FasL at the protein level on the surface of antigen- presenting cells (APCs) and vascular endothelium within 1 hour. In preliminary experiments, we demonstrated that delivery of FasL on donor APCs blocked alloreactive responses in naive and presensitized animals and prevented islet allograft rejection. Expression of FasL protein on vascular endothelium under conditions adapted from clinical settings prolonged survival of cardiac allografts. In this proposal, rats and mice will be immunized with allogeneic APCs expressing FasL at various times pre-and post- transplantation. FasL will also be expressed on the surface of heart endothelium for immune evasion and prevention of rejection. Several mutant and transgenic animals will be used to test whether apoptosis induced by FasL is the main mechanism of the observed immune nonresponsiveness. This protein-based approach may be readily applied to the clinic and may provide a significant advantage because of its safety and simplicity as compared with immunomodulatory approaches using DNA-based expression.